Equine infectious anemia virus and human immunodeficiency virus DNA synthesis in vitro: characterization of the endogenous reverse transcriptase reaction

Abstract

The endogenous reverse transcriptase reaction of equine infectious anemia virus (EIAV) has been studied, and conditions allowing synthesis of full-length minus-strand DNA have been determined. In contrast to results reported for other retroviruses, synthesis of EIAV full-length minus-strand DNA was not impaired by high concentrations of Nonidet P-40, a nonionic detergent used to make the virion envelope permeable. All components of the reaction were titrated for maximum synthesis of complete minus strands, and a time course under the standardized conditions was determined. Minor subgenomic bands were observed in some cases, and both the size and proportion varied with reaction conditions. Conditions established for full-length EIAV DNA synthesis also allowed full-genome-length human immunodeficiency virus type 1 DNA synthesis. The human immunodeficiency virus type 1 DNA product contained a greater proportion of reverse transcripts that were shorter than the complete virus genome. Also in contrast to EIAV, the endogenous synthesis of high-molecularweight human immunodeficiency virus type 1 DNA was drastically reduced at Nonidet P-40 concentrations above 0.02%. These results indicated that a detergent-stable core is not a property shared by all lentiviruses. The EIAV virion synthetic machinery is unusually stable and provides a convenient system for further in vitro study of reverse transcription.

Role of Vif in human immunodeficiency virus type 1 reverse transcription

“…Wild-type virions produced in CEM cells synthesized DNA products which ranged in size from 0.5 to 9.7 kb. Most of the DNA products were subgenomic species of less than approximately 7 kb, consistent with a previous report (4). vif mutant virions produced in CEM cells demonstrated a significant reduction in viral DNA synthesis ( Fig.…”

“…The size markers are HindIII fragments of lambda DNA. The single strands of the denatured 6.6-kb marker fragment appear as a doublet (4). Similar results were observed in three independent experiments.…”

“…However, the majority of products are full-length minus strand and a discontinuous plus strand, similar to the forms detected in the cytoplasm early after infection (2,27). In HIV-1 virions, the DNA products contain a much greater proportion of reverse transcripts that are shorter than the complete viral genome, most likely because of the relative sensitivity of the HIV-1 virion core to detergents (4,8,42).…”

“…Agarose gel electrophoresis. Endogenous RT products were resuspended in 10 mM Tris-HCl (pH 8.0)-1 mM EDTA and denatured by alkaline hydrolysis in 0.1 M NaOH for 1 h at 37ЊC prior to electrophoresis in 1% agarose in Tris-borate-EDTA (TBE) running buffer (4). DNA molecular weight markers were denatured, run in parallel, and visualized with ethidium bromide.…”

“…Virions are permeabilized with a nonionic detergent or mellitin and then incubated with high concentrations of deoxynucleoside triphosphates (dNTPs), a divalent cation, buffer, and radiolabeled dNTP. The endogenous RT reaction products represent a mixture of single-and double-stranded DNA and RNA-DNA hybrids, with low-molecular-weight products arising from discontinuous synthesis of viral DNA (1,2,4,16,32). In murine and avian retroviruses, the synthesis of fully infectious double-stranded viral DNA in vitro has been demonstrated (1,16,21,32).…”